Microfluidic devices and methods of use

ABSTRACT

A microfluidic device comprises pumps, valves, and fluid oscillation dampers. In a device employed for sorting, an entity is flowed by the pump along a flow channel through a detection region to a junction. Based upon an identity of the entity determined in the detection region, a waste or collection valve located on opposite branches of the flow channel at the junction are actuated, thereby routing the entity to either a waste pool or a collection pool. A damper structure may be located between the pump and the junction. The damper reduces the amplitude of oscillation pressure in the flow channel due to operation of the pump, thereby lessening oscillation in velocity of the entity during sorting process. The microfluidic device may be formed in a block of elastomer material, with thin membranes of the elastomer material deflectable into the flow channel to provide pump or valve functionality.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The instant nonprovisional patent application claims priority from U.S.Provisional Patent Application No. 60/237,938, entitled MICROFLUIDICDEVICES AND METHODS OF USE, filed Oct. 3, 2000. The instantnonprovisional patent application also claims priority from U.S.Provisional Patent Application No. 60/237,937, entitled VELOCITYINDEPENDENT MICROFLUIDIC FLOW CYTOMETRY, filed Oct. 3, 2000. Theseprovisional patent applications are incorporated by reference herein forall purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Work described herein has been supported, in part, by National Instituteof Health grant HG-01642-02. The United States Government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

Sorting of objects based upon size is extremely useful in many technicalfields. For example, many assays in biology require determination of thesize of molecular-sized entities. Of particular importance is themeasurement of length distribution of DNA molecules in a heterogeneoussolution. This is commonly done using gel electrophoresis, in which themolecules are separated by their differing mobility in a gel matrix inan applied electric field, and their positions detected by absorption oremission of radiation. The lengths of the DNA molecules are theninferred from their mobility.

While powerful, electrophoretic methods pose disadvantages. For mediumto large DNA molecules, resolution, i.e. the minimum length differenceat which different molecular lengths may be distinguished, is limited toapproximately 10% of the total length. For extremely large DNAmolecules, the conventional sorting procedure is not workable. Moreover,gel electrophoresis is a relatively lengthy procedure, and may requireon the order of hours or days to perform.

The sorting of cellular-sized entities is also an important task.Conventional flow cell sorters are designed to have a flow chamber witha nozzle and are based on the principle of hydrodynamic focusing withsheath flow. Most conventional cell sorters combine the technology ofpiezo-electric drop generation and electrostatic deflection to achievedroplet generation and high sorting rates. However, this approach offerssome important disadvantages. One disadvantage is that the complexity,size, and expense of the sorting device requires that it be reusable inorder to be cost-effective. Reuse can in turn lead to problems withresidual materials causing contamination of samples and turbulent fluidflow.

Another disadvantage of conventional sorting technologies is aninability to readily integrate sorting with other activities. Forexample, due to the mechanical complexity of conventional sortingapparatuses, pre-sorting activities such as labeling and post-sortingactivities such as crystallization are typically performed on differentdevices, requiring physical transfer of the pre- and post-sorted sampleto the sampling apparatus.

This transfer requires precise and careful handling in order to preventany loss of the frequently small volumes of sample involved. Moreover,sample handling for conventional sorter structures is time-consuming.The resulting delay may hinder analysis of materials having limitedlifetimes, or prevent sorting that is based upon time-dependentcriteria.

Therefore, there is a need in the art for a simple, inexpensive, andeasily fabricated integratable sorting device which relies upon themechanical control of fluid flow rather than upon electrical interactionbetween the particle and the solute.

SUMMARY OF THE INVENTION

An embodiment of a microfluidic device in accordance with the presentinvention comprises pumps, valves, and fluid oscillation dampers. In adevice employed for sorting, an entity is flowed by the pump along aflow channel through a detection region to a junction. Based upon anidentity of the entity determined in the detection region, a waste orcollection valve located on opposite branches of the flow channel isactuated, thereby routing the entity to either a waste pool or acollection pool. A damper structure may be located between the pump andthe junction. The damper reduces the amplitude of oscillation pressurein the flow channel due to operation of the pump, thereby lesseningoscillation in velocity of the entity during sorting process. Themicrofluidic device may be formed in a block of elastomer material, withthin membranes of the elastomer material deflectable into the flowchannel to provide pump or valve functionality.

An embodiment of a microfluidic device in accordance with the presentinvention comprises a flow channel, a pump operatively interconnected tosaid flow channel for moving a fluid in said flow channel, and a damperoperatively interconnected to said flow channel for reducing the fluidoscillation in said flow channel.

An embodiment of a microfluidic sorting device comprises a first flowchannel formed in a first layer of elastomer material, a first end ofthe first flow channel in fluid communication with a collection pool anda second end of the first flow channel in fluid communication with awaste pool. A second flow channel is formed in the first elastomerlayer, a first end of the second flow channel in fluid communicationwith an injection pool and a second end of the second flow channel influid communication with the first flow channel at a junction. Acollection valve is adjacent to a first side of the junction proximateto the collection pool, the collection valve comprising a first recessformed in a second elastomer layer overlying the first elastomer layer,the first recess separated from the first flow channel by a firstmembrane portion of the second elastomer layer deflectable into thefirst flow channel. A waste valve is adjacent to a second side of thejunction proximate to the waste pool, the waste valve comprising asecond recess formed in the second elastomer layer separated from thesecond flow channel by a second membrane portion of the second elastomerlayer deflectable into the first flow channel. A pump adjacent to athird side of the junction proximate to the injection pool, the pumpcomprising at least three pressure channels formed in the secondelastomer layer and separated from second flow channel by third membraneportions of the second elastomer layer deflectable into the second flowchannel. A detection region is positioned between the injection pool andthe junction, one of an open and closed state of the collection valveand the waste valve determined by an identity of a sortable entitydetected in the detection region.

An embodiment of a damper in accordance with the present invention for amicrofluidic device comprises a flow channel formed in an elastomermaterial, and an energy absorber adjacent to the flow channel andconfigured to absorb an energy of oscillation of a fluid positionedwithin the flow channel.

An embodiment of a sorting method in accordance with the presentinvention comprises deflecting a first elastomer membrane of anelastomer block into a flow channel to cause a sortable entity to flowinto a detection region positioned upstream of a junction in the flowchannel. The detection region is interrogated to identify the sortableentity within the detection region. Based upon an identity of thesortable entity, one of a second membrane and a third membrane of theelastomer block are deflected into one of a first branch flow channelportion and a second branch flow channel portion respectively, locateddownstream of the junction. This causes the sortable entity to flow toone of a collection pool or a waste pool.

An embodiment of a method for dampening pressure oscillations in a flowchannel comprises providing an energy absorber adjacent to the flowchannel, such that the energy absorber experiences a change in responsethe pressure oscillations.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIGS. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17A is a plan view of a flow layer of an addressable reactionchamber structure.

FIG. 17B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

FIG. 17C is an exploded perspective view of the addressable reactionchamber structure formed by bonding the control channel layer of FIG.17B to the top of the flow layer of FIG. 17A.

FIG. 17D is a sectional elevation view corresponding to FIG. 17C, takenalong line 28D-28D in FIG. 17C.

FIG. 18 is a schematic of a system adapted to selectively direct fluidflow into any of an array of reaction wells.

FIG. 19 is a schematic of a system adapted for selectable lateral flowbetween parallel flow channels.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIGS. 21A-21J show views of one embodiment of a normally-closed valvestructure in accordance with the present invention.

FIGS. 22A and 22B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention.

FIG. 24 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIG. 25 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIG. 26 is a cross-sectional view of one embodiment of a damperstructure in accordance with the present invention.

FIG. 27 is a cross-sectional view of an alternative embodiment of adamper structure in accordance with the present invention.

FIG. 28 is a plan view of another alternative embodiment of a damperstructure in accordance with the present invention.

FIGS. 29A-B are cross-sectional views of operation of one embodiment ofa circulation apparatus including a damper structure in accordance withthe present invention.

FIG. 30A is a schematic plan view of a cell sorter structure inaccordance with one embodiment of the present invention.

FIG. 30B is a photograph of a plan view of the cell sorter shown in FIG.30A.

FIGS. 31A and 31B are schematic views of a T-junction of a sorterstructure in accordance with an embodiment of the present inventionengaged in reversible sorting.

FIG. 32A plots cell velocity versus pump frequency for one embodiment ofa cell sorter in accordance with the present invention.

FIG. 32B plots mean reversible time versus pump frequency for theembodiment of a cell sorter of FIG. 32A.

FIG. 33A plots optical intensity over time for the cell sorter structureof FIG. 33C operated at first frequency.

FIG. 33B plots optical intensity over time for the cell sorter structureof FIG. 33C operated at first frequency.

FIG. 33C is a schematic view of a cell sorter structure in accordancewith an alternative embodiment in accordance with the present invention.

FIG. 34 plots flow velocity versus pump frequency for cell sortersfabricated from different elastomeric materials.

DESCRIPTION OF SPECIFIC EMBODIMENTS

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent applications Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No.09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filed Jun. 27,2000. These patent applications are hereby incorporated by reference.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIG. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIG. 7A and 7B, flow channels 30 and 32 are preferablydisposed at an angle to one another with a small membrane 25 ofsubstrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Dow Corning Sylgard 182, 184 or 186, or aliphaticurethane diacrylates such as (but not limited to) Ebecryl 270 or Irr 245from UCB Chemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from Dow Coming Sylgard 184. Thelayer containing the flow channels had an A:B ratio of 20:1 was spincoated at 5000 rpm. The layer containing the control channels had an A:Bratio of 5:1

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from pure acrylatedUrethane Ebe 270. A thin bottom layer was spin coated at 8000 rpm for 15seconds at 170° C. The top and bottom layers were initially cured underultraviolet light for 10 minutes under nitrogen utilizing a Model ELC500 device manufactured by Electrolite corporation. The assembled layerswere then cured for an additional 30 minutes. Reaction was catalyzed bya 0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-GeigyChemicals. The resulting elastomeric material exhibited moderateelasticity and adhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly(dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Bonding together of successive elastomer layers in accordance withembodiments of the present invention need not result in a monolithicelastomeric structure. Successive layers of different elastomermaterials can be bonded together to form an embodiment of a structure inaccordance with the present invention. For example General Electric RTVand Dow Corning Sylgard 184 can be bonded together to form a multilayerstructure.

Moreover, bonding together of successive elastomer layers in accordancewith the present invention need not be permanent. In certainembodiments, the strength of the bond between elastomer layers need onlymaintain contact and resist separation under the forces encounteredduring membrane actuation. Application of greater force will cause theelastomer layers to separate, for example allowing flushing and reuse offlow or control channels present in the separated layers.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

For example, a diluent may be included during formation of the elastomermaterial to alter its properties. In one embodiment, a diluent is addedto the elastomer comprising the membrane layer to lessen the stiffnessof the membrane and thereby reduce the actuation force required. Twoexamples of diluent for elastomer materials are General Electric SF96,and DMV-V21 manufactured by Gelest, Inc. of Tullytown, Pa. In general,the diluent is mixed with the elastomer at a ratio of between about 15%and 30%.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, polybutadiene, polychloroprene:

-   -   Polyisoprene, polybutadiene, and polychloroprene are all        polymerized from diene monomers, and therefore have one double        bond per monomer when polymerized. This double bond allows the        polymers to be converted to elastomers by vulcanization        (essentially, sulfur is used to form crosslinks between the        double bonds by heating). This would easily allow homogeneous        multilayer soft lithography by incomplete vulcanization of the        layers to be bonded; photoresist encapsulation would be possible        by a similar mechanism.        Polyisobutylene:    -   Pure polyisobutylene has no double bonds, but is crosslinked to        use as an elastomer by including a small amount (˜1%) of        isoprene in the polymerization. The isoprene monomers give        pendant double bonds on the polyisobutylene backbone, which may        then be vulcanized as above.        Poly(styrene-butadiene-styrene):    -   Poly(styrene-butadiene-styrene) is produced by living anionic        polymerization (that is, there is no natural chain-terminating        step in the reaction), so “live” polymer ends can exist in the        cured polymer. This makes it a natural candidate for the present        photoresist encapsulation system (where there will be plenty of        unreacted monomer in the liquid layer poured on top of the cured        layer). Incomplete curing would allow homogeneous multilayer        soft lithography (A to A bonding). The chemistry also        facilitates making one layer with extra butadiene (“A”) and        coupling agent and the other layer (“B”) with a butadiene        deficit (for heterogeneous multilayer soft lithography). SBS is        a “thermoset elastomer”, meaning that above a certain        temperature it melts and becomes plastic (as opposed to        elastic); reducing the temperature yields the elastomer again.        Thus, layers can be bonded together by heating.        Polyurethanes:    -   Polyurethanes are produced from di-isocyanates (A-A) and        di-alcohols or di-amines (B-B); since there are a large variety        of di-isocyanates and di-alcohols/amines, the number of        different types of polyurethanes is huge. The A vs. B nature of        the polymers, however, would make them useful for heterogeneous        multilayer soft lithography just as RTV 615 is: by using excess        A-A in one layer and excess B-B in the other layer.        Silicones:    -   Silicone polymers probably have the greatest structural variety,        and almost certainly have the greatest number of commercially        available formulations. The vinyl-to-(Si—H) crosslinking of RTV        615 (which allows both heterogeneous multilayer soft lithography        and photoresist encapsulation) has already been discussed, but        this is only one of several crosslinking methods used in        silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³), where:   (1)

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.        Thus even in this extremely simplified expression, deflection of        an elastomeric membrane in response to a pressure will be a        function of: the length, width, and thickness of the membrane,        the flexibility of the membrane (Young's modulus), and the        applied actuation force. Because each of these parameters will        vary widely depending upon the actual dimensions and physical        composition of a particular elastomeric device in accordance        with the present invention, a wide range of membrane thicknesses        and elasticities, channel widths, and actuation forces are        contemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

In certain embodiments, it may be useful to apply both positive andnegative pressures to actuate the membrane. For example, where theelastomer material is relatively inflexible, an extremely rapid responsetime is desired, or the flow channel dimensions are small, it may beuseful to apply a positive pressure to a control channel actuate themembrane into the flow channel, followed by a negative pressure to causethe membrane to be displaced out of the flow channel.

Moreover, it may also be useful to cause movement of fluid through themicrofluidic device by the direct application of pressure to the flowchannel, such as the application of positive pressure directly to theflow channel inlet, or application of negative pressure directly to theflow channel outlet. Direct application of pressure alone can drive theflow of fluid within the microfluidic device, or direct pressure may beutilized in conjunction with actuation of membranes overlying the flowchannel. Pressure applied directly to the flow channel can also serve toalter the speed of movement of materials thought the flow channel asdesired.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τ_(close) is expected to besmaller than τ_(open). There is also a lag between the control signaland control pressure response, due to the limitations of the miniaturevalve used to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open)=3.63 ms, τ_(open)=1.88 ms,t_(close)=2.15 ms, τ_(close)=0.51 ms. If 3τ each are allowed for openingand closing, the valve runs comfortably at 75 Hz when filled withaqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 20, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Alternate Valve Actuation Techniques

In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

For the membrane electrode to be sufficiently conductive to supportelectrostatic actuation, but not so mechanically stiff so as to impedethe valve's motion, a sufficiently flexible electrode must be providedin or over membrane 25. Such an electrode may be provided by a thinmetallization layer, doping the polymer with conductive material, ormaking the surface layer out of a conductive material.

In an exemplary aspect, the electrode present at the deflecting membranecan be provided by a thin metallization layer which can be provided, forexample, by sputtering a thin layer of metal such as 20 nm of gold. Inaddition to the formation of a metallized membrane by sputtering, othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating are also available. Physicaltransfer of a metal layer to the surface of the elastomer is alsoavailable, for example by evaporating a metal onto a flat substrate towhich it adheres poorly, and then placing the elastomer onto the metaland peeling the metal off of the substrate.

A conductive electrode 70 may also be formed by depositing carbon black(i.e. Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS). Alternatively, the electrode70 may be formed by constructing the entire layer 20 out of elastomerdoped with conductive material (i.e. carbon black or finely dividedmetal particles). Yet further alternatively, the electrode may be formedby electrostatic deposition, or by a chemical reaction that producescarbon. In experiments conducted by the present inventors, conductivitywas shown to increase with carbon black concentration from 5.6×10⁻¹⁶ toabout 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is not required tomove, may be either a compliant electrode as described above, or aconventional electrode such as evaporated gold, a metal plate, or adoped semiconductor electrode.

Magnetic actuation of the flow channels can be achieved by fabricatingthe membrane separating the flow channels with a magneticallypolarizable material such as iron, or a permanently magnetized materialsuch as polarized NdFeB. In experiments conducted by the presentinventors, magnetic silicone was created by the addition of iron powder(about 1 um particle size), up to 20% iron by weight.

Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied inhomogenous magnetic field.

The magnetic field causing actuation of the membrane can be generated ina variety of ways. In one embodiment, the magnetic field is generated byan extremely small inductive coil formed in or proximate to theelastomer membrane. The actuation effect of such a magnetic coil wouldbe localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

It is also possible to actuate the device by causing a fluid flow in thecontrol channel based upon the application of thermal energy, either bythermal expansion or by production of gas from liquid. For example, inone alternative embodiment in accordance with the present invention, apocket of fluid (e.g. in a fluid-filled control channel) is positionedover the flow channel. Fluid in the pocket can be in communication witha temperature variation system, for example a heater. Thermal expansionof the fluid, or conversion of material from the liquid to the gasphase, could result in an increase in pressure, closing the adjacentflow channel. Subsequent cooling of the fluid would relieve pressure andpermit the flow channel to open.

8. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIG. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log₂n) controllines.

9. Selectively Addressable Reaction Chambers Along Flow Lines

In a further embodiment of the invention, illustrated in FIGS. 17A, 17B,17C and 17D, a system for selectively directing fluid flow into one moreof a plurality of reaction chambers disposed along a flow line isprovided.

FIG. 17A shows a top view of a flow channel 30 having a plurality ofreaction chambers 80A and 80B disposed therealong. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

FIG. 17B shows a bottom plan view of another elastomeric layer 110 withtwo control lines 32A and 32B each being generally narrow, but havingwide extending portions 33A and 33B formed as recesses therein.

As seen in the exploded view of FIG. 17C, and assembled view of FIG.17D, elastomeric layer 110 is placed over elastomeric layer 100. Layers100 and 110 are then bonded together, and the integrated system operatesto selectively direct fluid flow F (through flow channel 30) into eitheror both of reaction chambers 80A and 80B, as follows. Pressurization ofcontrol line 32A will cause the membrane 25 (i.e.: the thin portion ofelastomer layer 100 located below extending portion 33A and over regions82A of reaction chamber 80A) to become depressed, thereby shutting offfluid flow passage in regions 82A, effectively sealing reaction chamber80 from flow channel 30. As can also be seen, extending portion 33A iswider than the remainder of control line 32A. As such, pressurization ofcontrol line 32A will not result in control line 32A sealing flowchannel 30.

As can be appreciated, either or both of control lines 32A and 32B canbe actuated at once. When both control lines 32A and 32B are pressurizedtogether, sample flow in flow channel 30 will enter neither of reactionchambers 80A or 80B.

The concept of selectably controlling fluid introduction into variousaddressable reaction chambers disposed along a flow line (FIGS. 17A-D)can be combined with concept of selectably controlling fluid flowthrough one or more of a plurality of parallel flow lines (FIG. 16) toyield a system in which a fluid sample or samples can be can be sent toany particular reaction chamber in an array of reaction chambers. Anexample of such a system is provided in FIG. 18, in which parallelcontrol channels 32A, 32B and 32C with extending portions 34 (all shownin phantom) selectively direct fluid flows F1 and F2 into any of thearray of reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

In yet another novel embodiment, fluid passage between parallel flowchannels is possible. Referring to FIG. 19, either or both of controllines 32A or 32D can be depressurized such that fluid flow throughlateral passageways 35 (between parallel flow channels 30A and 30B) ispermitted. In this aspect of the invention, pressurization of controllines 32C and 32D would shut flow channel 30A between 35A and 35B, andwould also shut lateral passageways 35B. As such, flow entering as flowF1 would sequentially travel through 30A, 35A and leave 30B as flow F4.

10. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIGS. 20 allows a switchable flow array to beconstructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

11. Normally-Closed Valve Structure

FIGS. 7B and 7H above depict a valve structure in which the elastomericmembrane is moveable from a first relaxed position to a second actuatedposition in which the flow channel is blocked. However, the presentinvention is not limited to this particular valve configuration.

FIGS. 21A-21J show a variety of views of a normally-closed valvestructure in which the elastomeric membrane is moveable from a firstrelaxed position blocking a flow channel, to a second actuated positionin which the flow channel is open, utilizing a negative controlpressure.

FIG. 21A shows a plan view, and FIG. 21B shows a cross sectional viewalong line 42B-42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 42B, in its relaxed, unactuated position,separating portion 4008 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

FIG. 21C shows a cross-sectional view of valve 4200 wherein separatingportion 4208 is in an actuated position. When the pressure withincontrol channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

The behavior of the membrane in response to an actuation force may bechanged by varying the width of the overlying control channel.Accordingly, FIGS. 21D-42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIG. 21E-F along line 42E-42E′ of FIG.21D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

FIGS. 21G and H show a cross-sectional views along line 40G-40G′ of FIG.21D. In comparison with the unactuated valve configuration shown in FIG.21G, FIG. 21H shows that reduced pressure within wider control channel4207 may under certain circumstances have the unwanted effect of pullingunderlying elastomer 4206 away from substrate 4205, thereby creatingundesirable void 4212.

Accordingly, FIG. 21I shows a plan view, and FIG. 21J shows across-sectional view along line 21J-21J′ of FIG. 21I, of valve structure4220 which avoids this problem by featuring control line 4204 with aminimum width except in segment 4204 a overlapping separating portion4208. As shown in FIG. 21J, even under actuated conditions the narrowercross-section of control channel 4204 reduces the attractive force onthe underlying elastomer material 4206, thereby preventing thiselastomer material from being drawn away from substrate 4205 andcreating an undesirable void.

While a normally-closed valve structure actuated in response to pressureis shown in FIGS. 21A-21J, a normally-closed valve in accordance withthe present invention is not limited to this configuration. For example,the separating portion obstructing the flow channel could alternativelybe manipulated by electric or magnetic fields, as described extensivelyabove.

12. Side-Actuated Valve

While the above description has focused upon microfabricated elastomericvalve structures in which a control channel is positioned above andseparated by an intervening elastomeric membrane from an underlying flowchannel, the present invention is not limited to this configuration.FIGS. 22A and 22B show plan views of one embodiment of a side-actuatedvalve structure in accordance with one embodiment of the presentinvention.

FIG. 22A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

FIG. 22B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

While a side-actuated valve structure actuated in response to pressureis shown in FIGS. 22A and 22B, a side-actuated valve in accordance withthe present invention is not limited to this configuration. For example,the elastomeric membrane portion located between the abutting flow andcontrol channels could alternatively be manipulated by electric ormagnetic fields, as described extensively above.

13. Composite Structures

Microfabricated elastomeric structures of the present invention may becombined with non-elastomeric materials to create composite structures.FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention. FIG. 23 showscomposite valve structure 5700 including first, thin elastomer layer5702 overlying semiconductor-type substrate 5704 having channel 5706formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

FIG. 24 shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

The structures shown in FIGS. 23 or 24 may be fabricated utilizingeither the multilayer soft lithography or encapsulation techniquesdescribed above. In the multilayer soft lithography method, theelastomer layer(s) would be formed and then placed over thesemiconductor substrate bearing the channel. In the encapsulationmethod, the channel would be first formed in the semiconductorsubstrate, and then the channel would be filled with a sacrificialmaterial such as photoresist. The elastomer would then be formed inplace over the substrate, with removal of the sacrificial materialproducing the channel overlaid by the elastomer membrane. As isdiscussed in detail below in connection with bonding of elastomer toother types of materials, the encapsulation approach may result in astronger seal between the elastomer membrane component and theunderlying nonelastomer substrate component.

As shown in FIGS. 23 and 24, a composite structure in accordance withembodiments of the present invention may include a hard substrate thatbears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 25, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

Many types of active structures may be present in the nonelastomersubstrate. Active structures that could be present in an underlying hardsubstrate include, but are not limited to, resistors, capacitors,photodiodes, transistors, chemical field effect transistors (chemFET's), amperometric/coulometric electrochemical sensors, fiber optics,fiber optic interconnects, light emitting diodes, laser diodes, verticalcavity surface emitting lasers (VCSEL's), micromirrors, accelerometers,pressure sensors, flow sensors, CMOS imaging arrays, CCD cameras,electronic logic, microprocessors, thermistors, Peltier coolers,waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

As is well known in the art, a vast variety of technologies can beutilized to fabricate active features in semiconductor and other typesof hard substrates, including but not limited printed circuit board(PCB) technology, CMOS, surface micromachining, bulk micromachining,printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

A first approach is to rely upon the simple hermetic seal resulting fromVan der Waals bonds formed when a substantially planar elastomer layeris placed into contact with a substantially planar layer of a harder,non-elastomer material. In one embodiment, bonding of RTV elastomer to aglass substrate created a composite structure capable of withstanding upto about 3-4 psi of pressure. This may be sufficient for many potentialapplications.

A second approach is to utilize a liquid layer to assist in bonding. Oneexample of this involves bonding elastomer to a hard glass substrate,wherein a weakly acidic solution (5 μl HCl in H₂O, pH 2) was applied toa glass substrate. The elastomer component was then placed into contactwith the glass substrate, and the composite structure baked at 37° C. toremove the water. This resulted in a bond between elastomer andnon-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

Exposure to ethanol can also cause device components to adhere together.In one embodiment, an RTV elastomer material and a glass substrate werewashed with ethanol and then dried under Nitrogen. The RTV elastomer wasthen placed into contact with the glass and the combination baked for 3hours at 80° C. Optionally, the RTV may also be exposed to a vacuum toremove any air bubbles trapped between the slice and the RTV. Thestrength of the adhesion between elastomer and glass using this methodhas withstood pressures in excess of 35 psi. The adhesion created usingthis method is not permanent, and the elastomer may be peeled off of theglass, washed, and resealed against the glass. This ethanol washingapproach can also be employed used to cause successive layers ofelastomer to bond together with sufficient strength to resist a pressureof 30 psi. In alternative embodiments, chemicals such as other alcoholsor diols could be used to promote adhesion between layers.

An embodiment of a method of promoting adhesion between layers of amicrofabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

A third approach is to create a covalent chemical bond between theelastomer component and functional groups introduced onto the surface ofa nonelastomer component. Examples of derivitization of a nonelastomersubstrate surface to produce such functional groups include exposing aglass substrate to agents such as vinyl silane or aminopropyltriethoxysilane (APTES), which may be useful to allow bonding of the glass tosilicone elastomer and polyurethane elastomer materials, respectively.

A fourth approach is to create a covalent chemical bond between theelastomer component and a functional group native to the surface of thenonelastomer component. For example, RTV elastomer can be created withan excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

II. Damper Structure

Embodiments of apparatuses and methods in accordance with the presentinvention are directed to microfluidic devices comprising pumps, valves,and fluid oscillation dampers. In this respect, the microfluidic devicesof the present invention is similar to that described in Unger et al.Science, 2000, 288, 113-116, which is incorporated herein by referencein its entirety. However, microfluidic devices of the present inventionmay further comprise a damper.

The advantages of microfluidic devices of the present invention includereduced fluid oscillation within a flow channel which reduces potentialvariability in detection means. As the fluid is pushed through the flowchannel by the pumps, there is a tendency for the fluid to oscillate,i.e., the fluid is pushed through the flow channel in a sinusoidalwave-like fashion. As this oscillating fluid passes through a detectorregion, different fluid depth passes through the detector region. Anddepending on a particular detection means used, this difference in fluiddepth can cause a higher “background noise” or an inaccurate reading bythe detector. By reducing or eliminating this fluid oscillation, the“background noise” is reduced and a more accurate reading by thedetector can be achieved.

Preferred devices are constructed by single and multilayer softlithography (MLSL) as detailed in commonly assigned U.S. patentapplication Ser. No. 09/605,520, filed Jun. 27, 2000, which isincorporated herein by reference in its entirety.

Microfluidic devices of the present invention comprise an integratedpump which can be electronic, magnetic, mechanical, or preferablypneumatic pumps. By using a pneumatic pump, microfluidic devices of thepresent invention allow more precise control of the fluid flow withinthe fluid channel. In addition, unlike electro-osmotic driven fluidflow, pneumatic pump allows the flow of fluids in both directions,thereby allowing reversible sorting of materials, as discussed ingreater detail below. Furthermore, a pneumatic pump provides at least 10times, preferably at least about 20 times, and more preferably at leastabout 30 times the fluid flow rate capacity compared to the capacity ofelectro-osmotic fluid flow.

In addition, microfluidic devices of the present invention may comprisea damper which reduces or eliminates the fluid oscillation within thefluid channel. The damper can any device which attenuates the fluidoscillation. For example, the damper can simply be a channel which isopen to the ambient atmosphere and has a thin elastic membrane betweenthe channel and the fluid flow channel. Preferably, the damper is anencapsulated pocket of fluid medium with a thin elastic membrane abovethe fluid flow channel. The fluid medium can be a liquid or, preferably,a gas. The damper is generally located above the fluid flow channelswith a thin membrane, preferably an elastic membrane, between the fluidflow channel and the damper. Typically, there is at least 1 damperposterior to the pump in the direction of the fluid flow. Preferably,there is at least 2 dampers, more preferably at least 3 dampers and mostpreferably at least about 5 dampers posterior to the pump.

The width of damper is at least as wide as the width of flow channelthat is located below the damper. In this manner, the entirecross-section of the flow channel is covered by the damper to ensureattenuation of fluid oscillation across the entire width of the flowchannel. Preferably, the width of damper is at least about 1.1 times thewidth of flow channel, more preferably at least about 1.3 times thewidth of the flow channel, and most preferably at least about 1.5 timesthe width of the flow channel. In this manner, the need for a precisealignment of the damper on top of the flow channel is eliminated.

The damper is separated from the fluid flow channel by a thin membrane.Preferably this thin membrane has sufficient elasticity to deflect“upward” when a fluid having a peak of sinusoidal wave-like passesunderneath. In this manner, some of the fluid oscillation energy isabsorbed by the damper, thereby reducing the height (i.e., peak) offluid oscillation. Typically, the thickness of the membrane between thedamper and the fluid flow channel depends on a variety of factorsincluding the depth and width of the flow channel, the amount of fluidoscillation produced by the pump and the elasticity (i.e., the material)of the membrane. One of ordinary skill in the art can readily determinethe proper membrane thickness to achieve a desired attenuation of fluidoscillation depending on a desired application and materials used.

In general, damper structures in accordance with embodiments of thepresent invention feature an energy absorber adjacent to the flowchannel, the energy absorber configured to experience a physical changein response to an oscillation within the flow channel. As describedbelow, the energy absorber can take several forms, including but notlimited to a flexible membrane, a pocket filled with a compressiblefluid, and/or flexible walls of the flow channel itself.

FIG. 26 shows a cross-sectional view of a first embodiment of a damperstructure in accordance with the present invention. Damper structure2600 comprises cavity 2602 separated from underlying flow channel 2604by membrane 2606 of elastomer layer 2608 in which cavity 2602 is formed.Oscillation in pressure 2610 in the fluid flowing through underlyingflow channel 2604 causes membrane 2606 to flex up and down, therebyabsorbing some of the energy of oscillation and providing for a moreuniform flow of material through channel 2604. The degree to whichdamper structure 2600 is capable of absorbing oscillation energy isdictated by a number of factors, including but not limited to, thelength of the flow channel covered by the membrane, the elasticity ofthe membrane, and the compressability of any liquid material present inthe cavity. The longer the membrane and the greater its flexion, thelarger amount of oscillation energy that can be absorbed. Moreover, asstated above, the damping effect can be amplified by positioning aseries of damper structures along the flow channel.

FIG. 27 shows a cross-sectional view of yet another embodiment of adamper structure in accordance with the present invention, whereindamper structure 2700 comprises portion 2702 a of flow channel 2702having a larger cross-section, an upper region of portion 2702 beingfilled with air or some other type of fluid 2704. As material 2706flowing through channel 2702 experiences pressure oscillations 2708 andeventually encounters damper 2700, energy is expended as material 2706pushes against fluid 2704. This expenditure of energy serves to reducethe amplitude of energy of oscillation of material in the flow channel.

FIG. 28 shows a plan view of yet another embodiment of a damperstructure in accordance with the present invention. Damper structure2800 comprises the combination of cavity 2802 overlying and separatedfrom underlying flow channel 2804 by membrane 2806, and constriction2808 in the width of flow channel 2804 positioned downstream ofcavity/membrane combination 2802/2806. In a manner analogous tooperation of a resistance-capacitance (RC) element of an electroniccircuit, cavity/membrane combination 2802/2806 serves as a flowcapacitor while constriction 2808 serves as a flow resistor, resultingin a reduction in amplitude of pressure oscillations 2810 downstream ofdamper structure 2800.

While the above embodiments have described dampers that are specificallyimplemented as separate structures in the architecture of a microfluidicdevice, embodiments of the present invention are not limited to suchstructures. For example, the elastomer material in which flow channelsare formed may itself serve to absorb pressure oscillations within theflow, independent of the presence of separate membrane/cavity structuresor fluid filled portions of enlarged flow channels. The damping effectof the elastomer material upon pressure oscillations would depend uponthe elasticity of the particular elastomer, and hence its ability tochange shape during absorption of energy from the oscillating flow.

Damper structures in accordance with embodiments of the presentinvention function reduce the amplitude of oscillations by absorbingenergy through displacement of a moveable portion of the damperstructure, for example a flexible membrane or a fiuid pocket. The damperstructures will generally operate most efficiently to reduce theamplitude of oscillations within a given frequency range. This frequencyrange is related to the speed of displacement and recovery of themoveable portion relative to the oscillation frequency. The speed ofdisplacement and recovery of the moveable element is in turn dictated bysuch factors as material composition, and the design and dimensions of aspecific damper structure.

Damper structures in accordance with embodiments of the presentinvention may also be utilized to create fluid circulationsubstructures. This is illustrated and described in conjunction withFIGS. 29A-29B, which show cross-sectional views of embodiments ofcirculation structures utilizing damper structures in accordance withthe present invention.

Fluid circulation system 2900 comprises flow channel 2902 formed inelastomer material 2904 and featuring valves 2904 and 2906 positioned ateither end. End valves 2904 and 2906 are actuated, such that elastomervalve membranes 2904 a and 2906 a project into and block flow channel2902, forming sealed flow channel segment 2902 a.

Pump 2908 and damper 2910 are positioned adjacent to sealed flow segment2902 a. Pump 2908 comprises recess 2908 a separated from underlying flowchannel 2902 by pump membrane 2908 b. Damper 2910 comprises cavity 2910a separated from underlying flow channel 2902 by damper membrane 2910 b.

As shown in FIGS. 29A-B, pump 2908 and damper 2910 cooperate to permit acontinuous circulation of fluid within sealed segment 2902 a.Specifically, pump 2908 is first actuated such that pump membrane 2908 bdeflects into flow channel 2902, increasing the pressure within sealedsegment 2902 a. In response to this increased pressure, damper membrane2910 b is displaced into cavity 2910 a and fluid within segment 2902 acirculates to occupy the additional space.

Subsequently, pump 2908 is deactuated such that pump membrane 2908 brelaxes to its original position, out of flow channel 2902. Because ofthe reduced pressure experienced by segment 2902 a as a result of thedeactuation of pump 2908, damper membrane 2910 b also relaxes back intoits original position, displacing material back into flow channel 2902.As a result of this action, the material within sealed segment 2902 aexperiences a back flow, and circulation is accomplished.

The circulation of material as just described may prove useful in anumber of applications. For example, where a mixture comprising severalcomponents is being manipulated by a microfluidic apparatus, thecirculation may serve to ensure homogeneity of the mixture. Similarly,where a suspension is being manipulated, the circulating action mayserve to maintain particles in suspension.

Moreover, certain components of a fluid being manipulated by amicrofluidic device, such as cellular material, may stick to flowchannel sidewalls. Maintenance of a continuous circulation within theflow channels may help prevent loss of material to the channel walls.

III. Sorting Applications

In one particular embodiment of the present invention, the microfluidicdevice comprises a T-channel for sorting materials (e.g., cells or largemolecules such as peptides, DNA's and other polymers) with fluid flowchannel dimensions of about 50 μm×35 μm (width×depth). The width ofpressure channels (i.e., pneumatic pump) and the damper is 100 μm and 80μm, respectively. The gap between the flow channel and the damper (orthe pressure channel) is about 5 to 6 μm. In order to produce such athin first layer, the MLSL process requires providing a layer of anelastomer (e.g., by spreading) which is typically thinner than mostother previously disclosed microfluidic devices. For example, when usingGE RTV 615 PDMS silicon rubber, previous microfluidic devices typicallyused 30:1 ratio of 615A:615B at 2000 rpm spin-coating to fabricate thefirst (i.e., bottom) layer of the elastomer and 3:1 ratio of A:B for thesecond elastomer layer. However, it has been found by the presentinventors that the silicon rubber does not cure when the ratio of 30:1is used in fabricating the above described dimensions of fluid flowchannels in the first elastomer layer.

Moreover, in order to produce a thin first elastomer layer, a higherspin-coating rate was required. For example, without using any diluent,GE RTV 615 PDMS silicon rubber A and B components in the ratio of about20:1 was required at 8000 rpm to produce the first elastomer layerhaving about 3.5 μm flow channel depth and about 5-6 μm thicknessbetween the flow channel and the damper (or the pressure channels). WhenSF-96 diluent was used, spin-coating at about 3000 rpm can be used toachieve a similar dimension first elastomer layer.

During fabrication of a mold, the photoresist is typically etched usinga mask, developed and heated. Heating of the developed photoresistreshapes trapezoid-shaped “ridges”, which ultimately form the channels,to a smooth rounded ridges and reduces the height of ridges from about20 μm to about 5 μm. This method, however, does not provide channelshaving depth of about 3.5 μm. The present inventors have found that thislimitation can be overcome by treating the developed photoresist withoxygen plasma (e.g., using SPI Plasma Prep II from SPI Supplies aDivision of Structure Probe, Inc., West Chester, Pa.) and heating thephotoresist at a lower heat setting. Unlike previous methods, where ahigher heat setting appear to chemically modify the photoresist, thelower heat setting used in the present invention does not chemicallyalter the photoresist.

Microfluidic devices of the present invention can be used in a varietyof applications such as sorting cells as disclosed in commonly assignedU.S. patent application Ser. No. 09/325,667 and the correspondingpublished PCT Application No. US99/13050, and sorting DNA's as disclosedin commonly assigned U.S. patent application Ser. No. 09/499,943, all ofwhich are incorporated herein by reference in their entirety.

The actual dimensions of a particular microfluidic device depend on itsapplication. For example, for sorting bacteria which typically have cellsize of about 1 μm, the width of fluid flow channel is generally in therange of from about 5 μm to about 50 μm and the depth of at least about5 μm. For sorting mammalian cells which have typically have cell size ofabout 30 μm, the width of fluid flow channel is generally in the rangeof from about 40 μm to about 60 μm and the depth of at least about 40μm. For DNA sorting, the dimensions of fluid flow channels can besignificantly less.

TABLE A below provides a nonexclusive, nonlimiting list of candidatesortable entities, their approximate size range, and the approximaterange of flow channel widths of a microfluidic apparatus at the point ofdetection of the entity. TABLE A APPROXIMATE SIZE APPROXIMATE RANGERANGE OF OF FLOW CHANNEL SORTABLE SORTABLE WIDTH AT DETECTION ENTITYENTITY (μm) POINT (μm) bacterial cell 1-10 5-50 mammalian cell  5-10010-500 egg cell  10-1000  10-1000 sperm cell 1-10 10-100 DNA strands0.003-1    0.1-10   proteins 0.01-1    1-10 micelles 0.1-100   1-500viruses 0.05-1    1-10 larvae 600-6500 VARIABLE beads 0.01-100  VARIABLE

The information presented in TABLE A is exemplary in nature, and isintended only as a nonexclusive listing of candidates for sortingutilizing embodiments in accordance with the present invention. Thesorting of other entities, and variation in approximate channel widthsutilized to sort the entities listed above, are possible and would varyaccording to the particular application.

One particular embodiment of the present invention is shown in FIGS. 30Aand 30B, where FIG. 30A is a schematic drawing of the microfluidicdevice shown in FIG. 30B. In this embodiment, the microfluidic devicecomprises an injection pool 3022, where a fluid containing a materialcan be introduced. The fluid is then pumped through the fluid flowchannel 3034 via a pneumatic pump 3010 which comprises three pressurechannels. By alternately pressurizing the three pressure channels, onecan pump the fluid through the fluid flow channel 3034 in a similarfashion to a peristaltic pump.

The fluid exiting the pump oscillates due to actions of the pump. Thefluid oscillation amplitude is attenuated by dampers 3014 which islocated above the flow channel 3034, behind the pump 3010 and before adetector (not shown). Initially, the collection valve 3018A is closedand the waste valve 3018B is open to allow the fluid to flow from theinjection pool 3022 through the T-junction 3038 and into the waste pool3030. When a desired material is detected by the detector (not shown),the waste valve 3018B is closed and the collection valve 3018A is openedto allow the material to be collected in the collection pool 3026. Thevalve 3018A and 3018B are interconnected to the detector through acomputer or other automated system to allow opening and closing ofappropriate valves depending on whether a desired material is detectedor not.

Dimensions of the embodiment shown above in FIGS. 30A and 30B are asfollows. The width of the peristaltic pumps is 100 μm. The width of thedampers is 80 μm. The width of the switch valves is 30 and 50 μm. Thedimensions of the T-channel is 50×3.5 μm. The dimensions at theT-junction are 5×3.5 μm. The thickness of the interlayer is 6 μm.

One such application for the microfluidic device described above is in areverse sorting of a material (e.g., beads, DNA's, peptides or otherpolymers, or cells) as shown in FIGS. 31A and 31B. In the reversesorting process, the material is allowed to flow towards the waste pool3030 as shown in FIG. 31A. When a desired material is detected by adetector (not shown) the pump (not shown) is reversed until the materialis again detected by the detector. At this point, the waste valve 3018Bis closed and the collection valve 3018A is opened, as shown in FIG.31B, and the flow of material is again reversed to allow the material toflow into the collection pool 3026. After which the collection valve3018A is closed and the waste valve 3018B is opened. This entire processis repeated until a desired amount of materials in the injection (orinput) pool 3022 is “sorted”. The reverse movement of materials in theflow channel as just described can be assisted by changing pressuresapplied directly to flow channel inlets and outlets.

In one embodiment of the present invention, E. Coli expressing GFP issorted using the reversible sorting process described above. As shown inFIGS. 32A and 32B, the cell velocity depends on the frequency of thepump. Thus, the cell velocity reaches a maximum of about 16 mm/sec atabout 100 Hz of pump rate. Moreover, as expected, the mean reverse timein FIG. 31B, which represents the time interval between detection of E.Coli expressing GFP, reversing the pump, and detection of the same E.Coli, decreases as the pump frequency is increased.

In another embodiment of the present invention provides sortingmaterials according to ratio of wavelengths (e.g., from laser inducedfluorescence). For example, by measuring two different fluorescencewavelengths (e.g., λ1 and λ2) and calculating the ratio of λ1 and λ2,one can determine a variety of information regarding the material, suchas the life cycle stage of cells, the stage of evolution of cells, thestrength of enzyme-substrate binding, the strength of drug interactionswith cells, receptors or enzymes, and other useful biological andnon-biological interactions.

Another embodiment of the present invention provides multipleinterrogation (i.e., observation or detection) of the same material atdifferent time intervals. For example, by closing of the valves 3018A or3018B in FIG. 33C and alternately pumping the fluid to and from theinput well 3022 at a particular intervals, the material can be made toflow to and from the input well 3022 through the detector (not shown).By oscillating this material through the detection window 3040, one canobserve the material at different time intervals. For example, a samplecan be interrogated at 10 Hz pump frequency as shown in FIG. 33A or at75 Hz pump frequency as shown in FIG. 33B. As expected, at a higher pumpfrequency, the material can be observed at shorter intervals. Suchobservation of materials at different time has variety of applicationsincluding monitoring cell developments, enzyme-substrate interactions,affect of drugs on a given cell or enzyme; measuring half-life of agiven material including drugs, compounds, polymers and the like; aswell as other biological applications.

The performance of a sorter structure in accordance with embodiments ofthe present invention may be dependent upon the elastomer materialutilized to fabricate the device. FIG. 34 plots flow velocity versuspump frequency for cell sorters fabricated from different elastomericmaterials, namely General Electric RTV 615 and Dow Coming Sylgard 184.

FIG. 34 shows that the flow velocity of cells through the cell sortersreached a maximum at a pumping frequency of about 50 Hz. The decline inflow velocity above this frequency may be attributable to incompleteopening and closing of the valves with each pumping cycle.

Moreover, different values for maximum pumping rates of the two sortingstructures are different. The RTV 615 cell sorter exhibits a maximumpumping rate of about 10,000 μm/sec, while the Sylgard 184 cell sorterexhibits a maximum pumping rate of about 14,000 μm/sec. Maximum flowrates of other elastomeric microfluidic devices in accordance with thepresent invention have ranged from about 6000 μm/sec to about 17,000μm/sec, but should be understood as merely exemplary and not limiting tothe scope of the present invention.

FIG. 34 indicates that the pumping rate of a cell sorter device may becontrolled by the identity, and hence flexibility, of the particularelastomer used. In the instant case, based upon the relative flexibilityof RTV 615 and Sylgard 184, the greater the elasticity of the elastomerresults in a faster rate of pumping.

Other changes, for example the addition of diluents to the elastomer orthe mixing of different ratios of A and B components of fluidic layer,may allow even further fine tuning of the pumping rate. Changing thedimensions of the fluidic channel may also allow tuning of the pumpingrate, as different volumes of fluid in the channels will be moved witheach actuation of the membrane.

While the sorting device described above utilizes a T-shaped junctionbetween flow channels, this is not required by the present invention.Other types of junctions, including but not limited to Y-shaped, or evenjunctions formed by the intersection of four or more flow channels,could be utilized for sorting and the device would remain within thescope of the present invention.

Moreover, while only a single sorting structure is illustrated above,the invention is not limited to this particular configuration. A sorterin accordance with embodiments of the present invention is readilyintegratable with other structures on the same microfabricated device.For example, embodiments in accordance with the present invention mayinclude a series of consecutively-arranged sorting structures useful forsegregating different components of a mixture through successive sortingoperations.

In addition, a microfluidic device in accordance with embodiments of thepresent invention could also include structures for pre-sorting andpost-sorting activities that are in direct fluid communication with thesorter. Examples of pre- or post-sorting activities that can beintegrated directly into a microfluidic device in accordance with thepresent invention include, but are not limited to, crystallization, celllysis, labeling, staining, filtering, separation, dialysis,chromatography, mixing, reaction, polymerase chain reaction, andincubation. Chambers and other structures for performing theseactivities can be integrated directly onto the microfabricatedelastomeric structure.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1.-9. (canceled)
 10. A microfluidic sorting device comprising: a firstflow channel formed in a first layer of elastomer material, a first endof the first flow channel in fluid communication with a collection pooland a second end of the first flow channel in fluid communication with awaste pool; a second flow channel formed in the first elastomer layer, afirst end of the second flow channel in fluid communication with aninjection pool and a second end of the second flow channel in fluidcommunication with the first flow channel at a junction; a collectionvalve adjacent to a first side of the junction proximate to thecollection pool, the collection valve comprising a first recess formedin a second elastomer layer overlying the first elastomer layer, thefirst recess separated from the first flow channel by a first membraneportion of the second elastomer layer deflectable into the first flowchannel; a waste valve adjacent to a second side of the junctionproximate to the waste pool, the waste valve comprising a second recessformed in the second elastomer layer separated from the second flowchannel by a second membrane portion of the second elastomer layerdeflectable into the first flow channel; a pump adjacent to a third sideof the junction proximate to the injection pool, the pump comprising atleast pressure channels formed in the second elastomer layer andseparated from second flow channel by third membrane portions of thesecond elastomer layer deflectable into the second flow channel; and adetection region positioned between the injection pool and the junction,one of an open and closed state of the collection valve and the wastevalve determined by an identity of a sortable entity detected in thedetection region.
 11. The microfluidic device of claim 10 furthercomprising a damper structure adjacent to the second flow channelbetween the pump and the detection region.
 12. The microfluidic deviceof claim 11 wherein the damper comprises a cavity formed in the secondelastomer layer and separated from the second flow channel by a flexiblemembrane, the flexible membrane deflectable into the cavity to absorbenergy in response to pressure oscillation within the second flowchannel, thereby reducing an amplitude of the pressure oscillation. 13.The microfluidic device of claim 12 further comprising a constriction ina width of the second flow channel between the flexible membrane and thejunction.
 14. The microfluidic device of claim 11 wherein the dampercomprises an enlarged portion of the flow channel partially filled witha fluid, the fluid compressible to absorb energy in response to pressureoscillation within the flow channel, thereby reducing an amplitude ofthe pressure oscillation.
 15. The microfluidic device of claim 10wherein the elastomer material forming walls of the first flow channelis deflectable to absorb energy in response to pressure oscillationwithin the flow channel, thereby reducing an amplitude of the pressureoscillation.
 16. The microfluidic device of claim 10 wherein thejunction is T-shaped.
 17. The microfluidic device of claim 10 furthercomprising a second sorter structure positioned between the waste valveand the waste pool. 18.-33. (canceled)